Fig. 4

P5091 retarded cell proliferation and induced cell apoptosis and G2/M cell cycle arrest by degrading PLK1 protein. a DU145 and VCaP cells were treated with P5091 for 2 days. Cells were stained with crystal violet and then lysed with 1% SDS. Cell viability was assessed at OD570 nm by spectrometer. b DU145 and VCaP were treated with P5091 for 24 h. Cells were stained with FITC-Annexin V and propidium iodide (PI). The apoptotic cells were analyzed by flow cytometry. c DU145 or VCaP cells were treated with P5091 for 2 days. Cells were labeled with PI, and cell cycle status was analyzed by flow cytometry. d DU145 or VCaP cells were treated with P5091 for 2 days, and the DNA in nuclei was stained with DAPI. Scale bar, 20 μM. The percent of mitotic cells were assessed according to nuclei morphology. e DU145 and VCaP cells were treated with the USP7 inhibitor P5091 for 24 h, and the protein levels of USP7 and PLK1 were assessed by immunoblotting. f DU145 cells were treated with P5091 (1 μM) for 24 h, followed by continuous CHX (50 μg/mL) treatment for 1–4 h. PLK1 protein levels were analyzed by immunoblotting. PLK1 protein levels were calculated by grayscale analysis. g DU145 cells were treated with P5091 for 12 h, followed by treatment with 50 μM MG132 for 8 h. PLK1 protein was assessed by western blot. PLK1 protein levels were calculated by grayscale analysis